**3. Preventive treatments and strategies to mitigate white wine unstable proteins**

#### **3.1 Effect of growing and harvest conditions on wine protein composition**

By using principal component analysis and clustering techniques, Sarmento et al. [32] pointed out that the most important factor affecting wine protein profile was the grape variety, and the growing region, whereas vinification practice

#### *White Wine Protein Instability: Origin, Preventive and Removal Strategies DOI: http://dx.doi.org/10.5772/intechopen.101713*

(industrial and laboratory scale) on the same varietal wine did not show a major effect. In grapevines, the synthesis of the PR proteins is regulated in a developmental and tissue-specific manner and occurs predominantly in the skins of the grapes [36, 57]. In *V. vinifera* cv. Muscat Gordo Blanco, both the concentration of the corresponding main thaumatin-like proteins and the berry-specific expression of the VvTL1 gene improved intensely after *véraison* and continued during grape maturation [58]. Identical developmental patterns were also found in the expression of genes encoding chitinases, some identical to those involved in wine protein haze [59–61]. Immunological research of *V. labruscana* cv. Concord likewise demonstrated that thaumatin-like proteins and chitinases accumulate during berry maturation [62]. PR proteins also exist in several other fruits such as banana [63], cherry [64] and kiwi fruit [58]. In all *V. vinifera* cultivars studied, thaumatin-like proteins and chitinases are the main soluble protein of grape berries [36, 58]. The prevalence of these PR proteins was evident at all phases of the grape berry growth next *véraison* [30]. Significantly, as the levels of extractable proteins in the grape berries continually rise during maturation, it can be supposed that the haze-forming potential growth as maturation continues [30, 58]. Pocock et al. [30] also showed that the increase of thaumatin-like proteins and chitinases initiated at berry softening for Muscat of Alexandria, Sultana, Shiraz grape varieties, Sauvignon Blanc and Pinot Noir grape varieties. As in healthy grape berries, PR protein synthesis seems to be caused by *véraison*, this does not signify that the traditional PR protein inducers, wounding, stress and pathogenic attack, cannot additionally modulate the grape berries' PR proteins concentration. These grape proteins in vitro display antifungal activity to *Botrytis cinerea*, *Uncinula necator*, *Phomopsis viticola*, *Elsinoe ampelina* and *Trichoderma harzianum* general fungal pathogens of grapevines [35, 62, 65–67]. The antifungal activity shown in vitro replicates the major function of the PR proteins *in vivo*, their expression in grapes afterward *veraison* represents a defence mechanism for grapes. Jayasankar et al. [66] give additional credibility to this hypothesis by indicating that after *in vitro* selection, grapevines regenerate with *E. ampelina* culture filtrates presented high constitutive expression of PR proteins, comprising VvTL1 and higher disease resistance. Works in which the PR proteins synthesis is changed by gene technology would permit us to explore this hypothesis more. Currently, there are slight chances that the wine turbidity problems could be resolved by decreasing the PR protein expression in grape berries as this could lead to the grapevine disease. In leaves and grape berries from infected grapevines with pathogens, improved expression of some PR genes and higher levels of some PR proteins have been shown [68–70]. In greenhouse experimentations, Monteiro et al. [67] showed in infected grape berries with *U. necator* augmented concentration of thaumatin-like proteins than in uninfected grape. Jacobs et al. [68] observed that in response to powdery mildew infection β-1,3-glucanase activity and chitinases augmented in leaves and grape berries, and that genes expression (VvGlub, VvChi3 and VvTL2), for coding PR proteins, was powerfully induced. Only VvTL2 of the three putative gene products has been found as a soluble protein in grape must and wines [13]. In Chardonnay *V. vinifera* cv. grape bunches, Girbau et al. [71] showed that occasioned powdery mildew infection augmented the concentration of a grape berry lesser thaumatinlike proteins, VvTL2, in wine. In infections with higher intensities (>30% of infected bunches), the wine turbidity values measured after a heat test were significantly higher. Marchal et al. [72] showed that grape must from infected grape berries by *B. cinerea* presented lower protein concentration, in opposing to expectations that fungal diseases would lead to higher concentration of PR proteins in grape, and suggested that proteolytic enzymes from *B. cinerea* were responsible for this. In culture media and on fruits such as apple, secretion of proteases by

*B. cinerea* has been observed [73] and in tomato [74]. Girbau et al. [71] also studied the influence of *B. cinerea* infected grapes on the vineyard and observed that infection resulted in noticeable reductions in the concentration of PR proteins in the grape berries. Similar although fewer tendencies of decreases in protein concentration were observed in laboratory experimentations in which otherwise healthy grape berries were inoculated with *B. cinerea* [71]. In this work even though these grapes were not vinified, the variance in protein concentration was predictable that between uninfected and infected grapes would also be shown in wines produced from uninfected and infected grapes. In the grape juice from *Botrytis*infected grape berries, the decrease in protein concentration did not appear to be an artefact of reduced extraction into juice due to desiccation or shrivelling of the fruits, nevertheless could be due to proteolytic degradation of grape PR proteins by enzymes of *B. cinerea* as suggested by Marchal et al. [72]. In grape must, protein concentrations were also decreased when in this medium *B. cinerea* was grown [71]. If these effects are due to the activity of proteolytic enzymes from *B. cinerea*, these enzymes have the capacity to substitute bentonite fining for protein stabilisation in oenology, an objective of many research efforts worldwide. The consequences of mechanical grape picking, a harvesting operation that could cause wounding, on the grape berries PR proteins concentration, are therefore of attention. Paetzold et al. [12] showed that grapes picked up by hand, originated grape must with lower protein concentration compared with that of mechanically harvested grapes. The absence of stalk throughout crushing led to lesser polyphenolic concentration in the grape juice compared with the grape juice from grapes picked up by hand, therefore fewer proteins were lost in complexes with phenolic compounds from grape juice from the fruit picked up mechanically. Dubourdieu and Canal-Llaubères [75] showed that wine produced with destalked grapes with maceration during 18 hours presented higher protein concentration than wine produced immediately by pressing of whole bunches. It was not elucidated, if this rise in protein concentration was due to the wounding of grapes that occurs during destalking or maceration or from the elimination of the grape stalks. Pocock and colleagues [36, 76] observed the influence of mechanical harvesting on the PR proteins in grapes and wine. Mechanical harvesting together with long transport of the grape berries leads to greater PR protein concentration in the grape juice and wine. Indeed, white grapes harvested mechanically, following transport was found to double the concentration of bentonite necessary for the avoidance of protein haze when compared with grape berries harvested by hand and transported from the same vineyard [76]. This does not seem to be a consequence of an increase in protein synthesis, as evaluations among hand picked up grapes, mechanical picked up intact grapes, and the major form of mechanical picked up grape berries—a combination of damaged grape berries and grape must—showed that few if any protein was formed as a consequence of stress provoked by mechanical grapes picked up. Protein concentration increase in grape must from mechanical picked up grape berries consequently look to be due to protein extraction from grape skins rather than a physiological wounding answer by the grapes. The influence of water stress established under some viticultural management practices has been studied, on the PR proteins expression in grape berries by determination of the PR protein concentration of *V. vinifera* cv. Shiraz grape berries in irrigation essays [30]. The absence of irrigation, did not lead to higher PR proteins concentration in the grape, however it provided a clear physiological marks of grapevine water stress. On a fixed quantity of protein per grape, it was observed that in the grape must from water stressed grape the protein content was greater than that from irrigated grape since grapes from irrigated grapevines were greater and thus grapes solutes were

#### *White Wine Protein Instability: Origin, Preventive and Removal Strategies DOI: http://dx.doi.org/10.5772/intechopen.101713*

fewer concentrated. The water stress influence on the grape dimension is an overall phenomenon [77] and it is probable that reports related to the wine turbidity problems, are higher in drought years and they are due many to a variation in the grape dimensions in these years instead of a direct physiological answer of the grapes to water stress in the formation of PR protein.
